Eukaryotic cells duplicate their DNA molecules during the S phase of the cell cycle. Bidirectional replication initiates at sites called "origins". Although some origins fire in early S phase and others in later S phase, each origin fires only once within a single S phase. Because regulated DNA replication is required for proper cell division, and because incorrectly regulated replication contributes to the genomic instability implicated in malignant progression, improved comprehension of regulation of DNA replication is likely to contribute to our understanding of cancer and other health conditions which involve cell division. Understanding the mechanisms by which eukaryotic DNA replication is regulated will require elucidation of the DNA sequences which specify origin function. Previous studies have revealed that origins in the budding yeast, Saccharomyces cerevisiae, consist, minimally, of a short sequence (the ARS consensus sequence, ACS) and a longer flanking sequence which is easily unwound but is otherwise variable from origin to origin (the DNA Unwinding Element, DUE). This combination of sequence elements is sufficient for origin activity in plasmids. Sequences permitting origin activity in plasmids are called ARS elements. In other eukaryotic organisms, nothing is known about the sequences required for origin function. This dearth of information is partly due to the fact that, in other tested eukaryotic organisms (mammalian cells and Drosophila), the commonly used origin mapping technique, two dimensional (2D) gel electrophoresis, provides results which suggest that replication initiates throughout a broad zone rather than at a precise site. In addition, it is difficult to test putative origins for function in Drosophila and mammalian cells; neither organism reproducibly supports autonomous plasmic replication (ARS function) or facile introduction of sequences into the genome by homologous recombination. We have just discovered a replication origin (the ura4 origin) in the fission yeast, Schizosaccharomyces pombe, and we have shown that it resembles origins in mammalian cells and Drosophila in the sense that 2D gel electrophoresis suggests that initiation occurs in a broad zone. However, unlike Drosophila and mammalian cells, S. pombe supports both ARS function and homologous recombination into the genome. In this application, we propose to utilize these experimental advantages to identify the minimal stretch of DNA required in cis for ura4 origin function (the minimal origin), to identify the important subsequences within the minimal origin, to use in vivo and chromatin footprinting techniques to identify protein-DNA interactions in the minimal origin region, and to purify and characterize proteins which bind to the minimal origin. Because of the resemblance between the ura4 origin and mammalian/Drosophila origins, we suspect that the data obtained will prove useful in developing an understanding of eukaryotic replication origins in general.